Animal venoms are complex mixtures of toxic components, including neurotoxins and cytotoxins with exquisite target specificity. Toxins with unique activities are of interest for drugs and neurophysiological research. Spiders, because of their venom complexity (200-1000 toxins in a single spider) and diversity (>44,500 described species!), likely contain the largest pool of toxin diversity of any venomous taxon, yet most major spider lineages are unexplored. Moreover, the field is in early stages of understanding evolutionary dynamics generating venom toxin diversity. With new availability of complete venom transcriptomes and the first spider genomes it is now possible to understand venom diversity and evolution at a new level of sophistication. Insight into evolutionary mechanisms influencing venoms can serve as a guide for toxin discovery and development of antivenom therapies. The goal of this project is to understand the phylogenetic scale at which there are significant differences in venom composition among spider taxa, and to infer mechanisms of evolution that contribute to these differences. This project continues comparative analyses of venomes in sicariid spiders and Haplogyne relatives. Sicariids include the notorious brown recluse whose bites cause dermonecrotic lesions and systemic effects in humans. There are ~130 described species and differences among them represent over ~120 million years of evolution since their most recent common ancestor. Evidence indicates the toxin responsible for dermonecrosis was recruited for venom function before the most recent common ancestor of the lineage. Sicariids are in the Haplogyne suborder within which, before this work, very little was known about venoms. Funded by NIH R15-GM-097696-01, cDNA transcriptomes for five sicariid taxa, three haplogynes and one outgroup were created and screened to produce nearly 5,000 venom gland transcripts. Combined with venom proteomics the minimal phylogenetic breadth of over 15 venom-expressed toxins was identified. However, these data are not comprehensive and missing toxins could reflect real absence, or artifacts of methods. There are three specific aims for this renewal: (1) to more thoroughly assess the phylogenetic distribution of venom toxin lineages across sicariids and haplogynes by adding next generation sequencing data; (2) to identify and analyze the gene families from which venom toxins were recruited by identifying non- venom expressed homologs in body transcriptomes and in the genome of Loxosceles reclusa; (3) to analyze evolutionary dynamics (duplications and selection) of individual lineages of toxins and compare them to identify parallel patterns in the evolution of toxins in the same venom functional complex. The toxins discovered in this work are a platform for discovery of natural components that contain unique neurophysiological activities. Preliminary data indicate discovery of divergent and unique toxins.